The present invention relates to improvements in therapy and prevention of conditions characterized by an elevated level of eosinophil leukocytes, i.e. conditions such as asthma and other chronic allergic diseases. More specifically, the present invention provides a method for down-regulating interleukin 5 (IL5) by enabling the production of antibodies against IL5 thereby reducing the level of activity of eosinophils. The invention also provides for methods of producing modified IL5 useful in this method as well as for the modified IL5 as such. Also encompassed by the present invention are nucleic acid fragments encoding modified IL5 as well as vectors incorporating these nucleic acid fragments and host cells and cell lines transformed therewith. The invention also provides for a method for the identification of IL5 analogues which are useful in the method of the invention as well as for compositions comprising modified IL5 or comprising nucleic acids encoding the IL5 analogues.
Asthma is a common disease of the airways, affecting about 10% of the population. The present treatments is primarily based on the administration of steroids and represents a market value exceeding well over a billion dollars. For yet unknown reasons the incidence and morbidity of asthmatics have increased worldwide over the past two decades. Today, an improved understanding of the immunological mechanisms involved in asthmatic conditions combined with an explosive development in biotechnology provides a new basis for the development of alternative and perhaps better strategies for treatment.
A general feature in the pathogenesis of asthma and other chronic allergic diseases has proven to be elevated numbers of eosinophils, especially in the bronchial mucosa of the lungs. Upon activation eosinophils secrete a number of mediators that are actively involved in the inflammatory airway response. In the activation of eosinophils, interleukin 5 (IL5) plays an important role.
IL5 is a cytokine found in many mammalian species and among others both the human and the murine gene for IL5 have been cloned (Tanabe et al., 1987, Campbell et al., 1988). The human gene consists of four exons with three introns positioned at chromosome 5 and codes for a 134 amino acid residue precursor, including a 19 amino acid N-terminal leader sequence which has the amino acid sequence set forth in SEQ ID NO: 62. Posttranslational cleavage generates the mature 115 amino acid residue protein (SEQ ID NO: 1). The murine IL5 (mIL5) gene similarly codes for a 133 amino acid residue pre-cursor with a 20 amino acid leader sequence which has the amino acid sequence set forth in SEQ ID NO: 64. The processed mature mIL5 is thus 113 amino acid residues long (SEQ ID NO: 12), missing two N-terminal amino acid residues by alignment with human IL5. The amino acid sequences of hIL5 and mIL5 are 70% identical compared to 77% at nucleotide level of the coding regions (Azuma et al., 1986). Higher similarity was reported within human primates; 99% identity is reported for the coding regions of the human and the Rhesus monkey nucleotide sequences (Villinger et al., 1995).
The human amino acid sequence has two potential N-glycosylation sites and the murine three. Human IL5 has been shown to be both N-glycosylated as well as O-glycosylated at Thr 3. Studies of hIL5 has demonstrated that the glycosylation is not necessary for the biological activity even though the stability seems to be affected by de-glycosylation (Tominaga et al., 1990; Kodama et al., 1993).
Structure of IL5
The active IL5 is a homo-dimer and the 3-dimensional structure of recombinant hIL5 has been determined by X-ray crystallography (Milburn et al., 1993). The 2 monomers are organised in an antiparallel manner and covalently bound by two interchain disulfide bridges (44-87xe2x80x2 and 87-44xe2x80x2), thus engaging all 4 cysteines of the 2 monomers.
The secondary structure of the monomers consists of 4 xcex1-helices (A-D) intermitted by 3 linking regions (loops) including two short stretches of xcex2-sheets. This 4xcex1 helix bundle is known as the xe2x80x9ccommon cytokine foldxe2x80x9d, which has also been reported for IL-2, IL-4, GM-CSF, and M-CSF. But all these are monomers and the homodimer-structure in which the D-helix completes the 4xcex1 helix motif of the opposite monomer is unique to IL5.
The native monomers alone has been shown to be biologically inactive (for reviews see Callard and Gearing, 1994; Takutsu et al., 1997). It is nevertheless possible to produce a modified recombinant biologically active monomer by inserting 8 additional amino acid residues in loop 3, connecting the helices C and D. This enables helix D to complete the 4 helix structure within one polypeptide chain and thus enable the monomer to interact with its receptor (Dickason and Huston, 1996; Dickason et al., 1996).
The IL5 receptor is primarily present on eosinophils and it is composed of an xcex1-chain and a xcex2-chain. The xcex1-chain of the receptor is specific for IL5 and the xcex2-chain, which assure high-affinity binding and signal transduction, is shared with the hetero-dimer receptors for IL-3 and GM-CSF. The sharing of a receptor component could be the reason for the cross-competition seen between IL5, IL-3 and GM-CSF (for review, see Lopez et al., 1992). However, it was recently demonstrated that the regulation of the IL5R is distinct from the regulation of the IL-3R and the GM-CSFR, further indicating a highly specialised role of IL5 in the regulation of the eosinophilic response (Wang et al., 1998).
The C-terminal part of IL5 seems to be important in both binding to the IL5R and for the biological activity, since removal of more than two C-terminal amino acid residues results in a decline in both the binding affinity to the IL5 R and in the biological activity in an IL5 bioassay (Proudfoot et al., 1996). Other residues have also been found to be important for binding to the receptor, such as Glu12, which is involved in binding to the xcex2-chain, while the Arg90 and Glu109 residues are involved in the binding to the xcex1-chain of the receptor. In general, binding to the IL5R seems to occur in regions overlapping helices A and D, where helix D is primarily responsible for the binding to the specific IL5R xcex1-chain (Graber et al., 1995; Takastsu et al., 1997).
IL5""s Homology to Other Proteins
The two 4-helix domain motifs seen in the homodimer has strikingly similar secondary and tertiary structure as compared to the cytokine fold found in GM-CSF and M-CSF, IL-2, IL-4 and human and porcine growth hormone (Milburn et al., 1993). However, even though striking similarities are also observed in the intron/exon organisation and position of cysteines (Tanabe et al., 1987; Cambell et al., 1988) suggesting a phylogenetic relationship with IL-2, IL-4 and GM-CSF, no significant homology with any of these or other cytokines is observed from the amino acid sequence.
Biological Activity of IL5
IL5 is mainly secreted by fully differentiated Th2 cells, mast cells and eosinophils (Cousins et al., 1994; Takutsu et al., 1997). It has been shown to act on eosinophils, basophiles, cytotoxic T lymphocytes and on murine B cells (Callard and Gearing, 1994; Takutsu et al., 1997). The effects of IL5 on human cells are still a matter of controversy. Augmentation of immunoglobulin synthesis under certain circumstances and binding to a variety of human B cell lines have been demonstrated. Even though mRNA for the hIL5R has been found in human B-cells, the actual presence of the receptor on these cells has still to be verified (Baumann and Paul, 1997; Huston et al., 1996).
The actions of IL5 on eosinophils include chemotaxis, enhanced adhesion to endothelial cells, activation and terminal differentiation of the cells. Furthermore it has been demonstrated that IL5 prevents mature eosinophils from apoptosis (Yamaguchi et al., 1991). These findings have contributed to the present concept of IL5 as being the most important cytokine for eosinophil differentiation (Corrigan and Kay, 1996; Karlen et al., 1998).
Physiologically, IL5 and its associated eosinophil activation is considered to serve a protective role against helminthic infections and possibly against certain tumours, since these diseases are typically accompanied by peripheral blood eosinophilia (Takutsu et al., 1997; Sanderson et al., 1992). It is, however, somewhat speculative as in two studies the authors failed to show any effect beside eosinophil down-regulating following administration of antibodies against IL5 on the immunity (e.g. IgE levels) against Nippostrongylus braziliensis or Schistosoma mansoni in mice infected with these parasites (Sher et al., 1990; Coffman et al., 1989).
IL5 Transgenic and xe2x80x9cKnock-outxe2x80x9d Animals
Studies of transgenic mice expressing IL5 or knock-out mice deficient for IL5 have given further knowledge of the physiological role of IL5.
Several IL5 transgenic mice have been reported:
A transgenic mouse expressing the IL5 gene in T cells was reported to have an increased white blood cell level characterised by expansion of B220+ B lymphocytes and profound eosinophilia. This was accompanied by a massive peritoneal cavity cell exudate dominated by eosinophils and infiltration of eosinophils in nearly all organ systems (Lee et al., 1997a).
Another transgenic mouse, expressing the IL5 gene under control of a metallothionin promoter was characterised by an increase in the serum levels of IgM and IgA, a massive eosinophilia in peripheral blood and many other organs accompanied by the expansion of a distinctive CD5+ B cell population, which produce auto-antibodies (Tominaga et al., 1991).
A third study involved a transgenic mouse constitutively expressing IL5 in the lungs. These animals developed pathophysiological changes resembling those of human asthma, including eosinophil invasion of peribronchial spaces, epithelial hypertrophy and increased mucus production. Furthermore, development of airway hyper responsiveness was seen in the absence of antigens (Lee et al., 1997b).
IL5-deficient mice (xe2x80x98knock-outxe2x80x99 mice) have also been studied. These mice (C57BL/6) have no obvious signs of disease and are fertile. The immunoglobulin levels and the specific antibody responses to DNP-OVA were normal. Basal levels of eosinophils are produced, but are 2-3 times lower than in control animals, indicating that eosinophils can be produced in the complete absence of IL5. When these mice were infected with Mesocestoides corti the eosinophilia normally seen was abolished and this absence of eosinophilia did not affect the worm burden produced by this parasite (Kopf et al., 1996).
In a study by Foster et al. (1996), the effect of IL5 knock-out on a common model of atopic airway inflammation was investigated. Sensitisation and aerosol challenge of mice with ovalbumin normally result in airway eosinophilia, airway hyperreactivity to xcex2-methacholin and extensive lung damage analogous to that seen in asthma. In the IL5 deficient mice the eosinophilia, airway hyperreactivity and lung damage were abolished. When IL5 expression in these mice was reconstituted, the aero-allergen induced eosinophilia and airway dysfunction were restored.
Pathophysiologic Role of IL5
Asthma affect about 10% of the population worldwide and for yet unknown reasons the incidence and morbidity have increased over the past two decades (Ortega and Busse, 1997). It is a chronic airway disease characterised by recurrent and usually reversible air flow obstruction, inflammation and hyper responsiveness (Moxam and Costello, 1990). This produces symptoms of wheezing and breathlessness, which in severe cases can be fatal.
The animal experiments referred to above using transgenic mice constitutively expressing IL5 in the lungs (Lee et al., 1997a) and the IL5 deficient xe2x80x9cknock-outxe2x80x9d mice (Foster et al., 1996) strongly implicate a crucial role of IL5 in the pathogenesis of asthma. Further evidence supporting this can be deduced from several studies including asthmatic individuals.
Eosinophilia has been identified in bronchoalveolar lavage (BAL) fluid and in bronchial mucosal biopsies of subjects with asthma and correlates with disease severity. Several eosinophil products have been identified in the BAL fluid of patients with asthma and numbers of peripheral blood eosinophils correlate with asthma severity (Ortega and Busse 1997).
IL5 serum concentration was found to be elevated (median concentration 150 pg/ml) in 15 out of 29 patients with chronic severe asthma as compared to control subjects (Alexander et al., 1994).
In another study involving both non-atopic and atopic asthmatics, it was found that an enhanced IL5 production by helper T cells seems to cause the eosinophilic inflammation of both atopic and non-atopic asthma (Mori et al., 1997).
Other results also indicate that IL5 has a distinct role in other atopic diseases. Allergen induced systemic episodes in individuals with allergic rhinitis has recently been shown to correlate to allergen induced IL5 synthesis rather than IgE (Ohashi et al., 1998). The correlation of atopic reactions is also demonstrated in a study by Barata et al. (1998) in which a significant expression of IL5 by T-cells in a cutaneous late phase reaction is demonstrated.
These and other results have led several authors as Corrigan and Kay (1996), Danzig and Cuss (1997) to identify and recommend IL5 as a primary target in the development of a better treatment for asthma and atopic diseases involving eosinophilic inflammation. Chronic tissue damaging hypereosinophilia induced by parasitic infection, topical pulmonary eosinophilia and hypereosinophilic syndrome are examples of other pathogenic conditions that could be addressed by IL5 down regulation.
In vivo Demonstration of the Role of IL5
In several studies with rodent models of asthma it has been shown that treatment with monoclonal antibodies against IL5 (anti-IL5 mAb) results in dose-related inhibition of eosinophilia, as compared to non-treated controls (Nagai et al., 1993a and b; Chand et al., 1992; Coeffier et al., 1994; Kung et al., 1995; Underwood et al., 1996). In the study by Nagai et al. (1993a) the effect was also observed by treating the sensitised Balb/c mice with soluble IL5 receptor xcex1.
In one study with Balb/c mice (Hamelmann et al., 1997) and four studies with guinea pigs it was additionally shown that anti-IL5 mAb could inhibit airway hyperreactivity elicited with various substances in antigen sensitised animals (Mauser et al., 1993; Akutsu et al., 1995; van Oosterhout et al., 1995 and 1993). In some of the studies beneficial effects (cf. table 1) of the anti-IL5 mAb treatment were also observed microscopically (Mauser et al., 1993; Akutsu et al., 1995; Kung et al., 1995). Importantly, in the study by Kung et al. (1995) a reduction of pulmonary inflammation in B6D2F1 mice was seen both when anti-IL5 mAb was administered hours before antigen challenge and also when administered up to five days after antigen challenge, indicating that the effect of anti-IL5 mAb may be both prophylactic and therapeutic for airway inflammation. This effect, however, was not observed by Underwood et al. when guinea pigs were given anti-IL5 mAb two hours after antigen challenge (Underwood et al., 1996).
In a study using a monkey model of asthma, Mauser et al. (1995) reported an inhibition of airway hyper reactivity after antigen challenge, when rat anti mouse-IL5 mAb was given 1 hour before antigen challenge. In addition, there was 75% reduction in the number of eosinophils in bronchoalveolar lavage (BAL) of antibody treated animals, as compared to non-treated controls. The effects on eosinophilia and hyperresponsiveness of anti-IL5 mAb was seen for up to three months after treatment (Mauser et al., 1995). Regarding allergic hyperresponsiveness, the results from studies by Nagai et al. (1993a and 1993b) document no reduction in hyperresponsiveness in conjunction to a reduction of eosinophil numbers in BAL.
All anti-IL5 mAb in vivo experiments mentioned so far have been done with rat-anti-mouse monoclonal antibodies. Egan et al. (1995) have reported experiments using humanised rat-anti-human IL5 monoclonal antibodies, called Sch 55700. These mAbs, inhibited lung lavage eosinophilia by 75% at a dose of 0,3 mg/kg when administered to sensitised monkeys. When Sch 55700 was given at 1 mg/kg in allergic mice, inhibition of airway eosinophilia was also observed.
Treatment of Asthma at Present and in the Future
The current treatment of asthma is, as mentioned, corticosteroids which, by their anti-inflammatory action, are the most powerful drugs. Besides this, xcex22 agonists and methyl xanthine derivatives which all cause bronchodilation, and disodium chromoglycate which xe2x80x98stabilisesxe2x80x99 mast cells, thereby preventing mediator release, all have proven beneficial in asthma patients (Ortega and Busse 1997).
Future treatment of asthma may as discussed above include anti-IL5 mAbs. Celltech in corporation with Schering Plough have anti-IL5 mAb in phase I clinical trial for treatment of asthma. However, treatment with monoclonal antibodies entails a number of drawbacks. First of all, the development and production costs for a safe mAB (e.g. a humanised mAB) are very high, resulting in an expensive therapeutic product for the end user. Second, mABs have the disadvantageous characteristic seen from a patient point of view that they have to be administered with relatively short intervals. Third, by nature mABs exhibit a narrow specificity against one single epitope of the antigen. And, finally, mABs (even humanised) are immunogenic, leading to an increasingly fast inactivation of administered antibodies as treatment progresses over time.
Also use of antisense IL5 oligonucleotides for antisense therapy has been suggested by the company Hybridon for the treatment of asthma, allergies and inflammation. However, the antisense technology has proven to be technically difficult and, in fact, conclusive evidence of the feasibility of antisense therapy in humans has not yet been established.
Finally, WO 97/45448 (Bresagen Limited/Medvet Science) proposes the use of xe2x80x9cmodified and variant forms of IL5 molecules capable of antagonising the activity of IL5xe2x80x9d in ameliorating, abating or otherwise reducing the aberrant effects caused by native or mutant forms of IL5. The antagonizing effect is reported to be the result of the variant forms of IL5 binding to the low affinity a chain of IL5R but not to the high affinity receptors; in this way the variants compete with IL5 for binding to its receptors without exerting the physiological effects of IL5.
Other atopic diseases involving eosinophilic inflammation are treated with either the symptomatica mentioned for asthma or immune therapy (IT) using hyposensitization with allergen extracts. The latter type of treatment is known to be effective against allergies against one or a few antigens, whereas IT is not feasible in the treatment of multiple allergies. Furthermore, the time scale for obtaining clinical improvement in patients susceptible to treatment is very long for conventional IT.
Thus, in spite of existing and possible future therapies for chronic allergic diseases such as asthma, there is a definite need for alternative ways of treating and ameliorating this and other chronic allergic diseases.
The object of the present invention is to provide novel therapies against chronic allergic conditions (such as asthma) characterized by eosinophilia. A further object is to develop an autovaccine against IL5, in order to obtain a novel treatment for asthma and for other pathological disorders involving chronic airway inflammation.
The T-cell derived cytokine IL5 has, as mentioned above, a crucial role in orchestrating the eosinophilic response, affecting both the production, the localisation and the activation of eosinophils. As IL5 has not otherwise been reported to have a central role in the development of a protective immune response, this particular cytokine is in the opinion of the inventors an attractive therapeutic target for the treatment of asthma.
The general aim according to the present invention is to decrease the pathogenic levels of eosinophils in the airways of the asthma patient by down-regulating of the IL5 levels, since eosinophils depend on IL5 for attraction and activation. The result of a decreased eosinophil number in the airway mucosa would be a concomitant decrease in the airway inflammation, corresponding to a clinical improvement in the asthmatic patient.
The potential effect of such an approach has already been demonstrated in studies using anti IL5 monoclonal antibodies in animal models of airway inflammation, cf. the xe2x80x9cPREAMBLE TO EXAMPLESxe2x80x9d.
This current invention, however, takes the results obtained through passive immunisation one step further by using the approach of generating an active immune response through the concept of autovaccination. To the best of the inventor""s knowledge, such an approach has never been suggested before.
The advantage of treating asthmatics with an IL5 autovaccine, as compared to current treatment with corticosteroids etc., is a reduction and/or elimination of side effects and most likely a better effect in terms of duration. When compared to anti-IL5 mAbs, the effect of an induced polyclonal Ab response is expected to be superior to passively injected monoclonal immunoglobulins since the polyclonal response has a broader specificity. Improvements with respect to administration regimen are also expected (since effective autovaccines described herein typically would require a maximum of 2-6 administrations per year).
When compared to hyposensitization, the present invention offers the attractive aspect of being non-specific; this is especially relevant when dealing with multi-allergic patients.
Thus, in its broadest and most general scope, the present invention relates to a method for in vivo down-regulation of interleukin 5 (IL5) activity in an animal, including a human being, the method comprising effecting presentation to the animal""s immune system of an immunologically effective amount of
at least one IL5 polypeptide or subsequence thereof which has been formulated so that immunization of the animal with the IL5 polypeptide or subsequence thereof induces production of antibodies against the IL5 polypeptide, and/or
at least one IL5 analogue wherein is introduced at least one modification in the IL5 amino acid sequence which has as a result that immunization of the animal with the analogue induces production of antibodies against the IL5 polypeptide.
The most attractive aspect of this approach is that e.g. asthma can be controlled by periodic but not very frequent immunizations, in contrast to a therapeutic approach which involves administration of anti-IL5 or molecules having a binding affinity to IL5 analogous therewith. It is expected that 1-4 annual injections with an immunogenic composition according to the invention will be sufficient to obtain the desired effect, whereas administration of other inhibitors of IL5 activity does or will require daily, or at least weekly, administrations.
The invention also relates to IL5 analogues as well as to nucleic acid fragments encoding a subset of these. Also immunogenic compositions comprising the analogues or the nucleic acid fragments are part of the invention.
The invention also relates to a method of identifying analogues of IL5 as well as a method for preparing a composition comprising the IL5 analogues.